T CELL ENGAGING BISPECIFIC ANTIBODIES GENERATED FROM A NOVEL, LARGE AND DIVERSE CD3 PANEL DEMONSTRATE FUNCTIONALITY UNCOUPLED FROM AFFINITY

1. A novel, large and diverse cLC CD3 Fab panel for TCE Biclonics® format providesopportunities to identify TCEs with optimal activity as TCE activity that cannot bepredicted from CD3 affinity alone:• CD3 affinity and CD3xTAA TCE activity can be dis-linked• CD3 induced lysis can be uncoupled from cytokine release• Activity of CD3 Fab can be tailored to specific TAA.

2. A broad therapeutic window for a TCE may be optimally achieved by empiricalevaluation of CD3 and TAA Fab arm panels in CD3xTAA IgG format using a highthroughput screening setup.

Pieter Fokko van Loo, Thèrese Visser, Katinka van Zoest, Abdul Basmeleh, Carina Bartelink, Roy Nijhuis, Karin de Cortie, Marina Kraaij, Henrike Veninga, John de Kruif, Mark Throsby | Merus N.V., Utrecht, The Netherlands

CONTACT INFORMATION

METHODS

INTRODUCTION

T cell engaging bispecific antibodies (TCEs) recruitand activate T cells to specifically lyse tumor cells.

TCEs have demonstrated clinically meaningfulresponses in different hematological malignanciesbut their clinical application may be limited bytoxicities associated with cytokine releasesyndrome (CRS).

TCEs could potentially be improved for therapeuticuse by utilizing CD3 binding domains thatoptimally balance proliferation, tumor cell killingand cytokine release of TCE-activated T cells.

Large and diverse anti-CD3 antibody panels arechallenging to generate.

Corresponding author: Pieter Fokko van Loo, Director Oncology ImmunologyEmail address: p.vanloo@merus.nl

We aimed to generate and functionally characterize a novel, large and diverse common lightchain CD3 antibody panel for the discovery and optimization of T cell engaging antibodies.

OBJECTIVE

RESULTS

• We generated a panel of more than 180 unique cLC anti-CD3 antibodies, derived fromMeMo®

• The CD3 panel comprised eight distinct groups, analysis predicts each CD3 Fab grouporiginates from a unique B cell clone.

• Anti-CD3 binding affinity for entire panel was evaluated in monovalent format (TAAxCD3Biclonics®) by flow cytometry using HBL-ALL cells and compared to monovalent bindingreference antibodies with known affinities

• The antibodies showed affinities for CD3 in the low pM to high nM range.

Large common light chain CD3 antibody panel

• We paired representative variants from each CD3 Fab group with a TAA Fab, tested theCD3xTAA Biclonics® for CD3 binding in flow cytometry, and evaluated them for their capacityto lyse target cells in a T cell cytotoxicity assay.

• Interestingly, Biclonics® from two groups bound to CD3 with different binding affinity to CD3showed however relatively equivalent cytolytic activity.

CD3 affinity and CD3xTAA Biclonics® activity are uncoupled

CD3 induced lysis and cytokine release can be uncoupled

• Several Biclonics® with variants within a CD3 Fab group had similar cytolysis butsignificantly lower IFN-γ and IL-6 release, demonstrating that CD3 induced lysis can beuncoupled from cytokine production.

The CD3 Fab panel and two Fabs targeting two tumorassociated antigens were formatted together as TAAxCD3full-length IgG1 human bispecific antibodies with Fceffector function silenced (TCE Biclonics®) and evaluatedin functional assays in which tumor cells were co-cultured with resting human T cells as effector cells.Biclonics® are bispecific human IgG1 moleculescomprising two cLC and two heavy chains with differentvariable regions. Charge engineering in the CH3 regionsdirects preferential bispecific heterodimer formation.

Optimal activity of CD3 Fab can be TAA specific

• Representative variants from four CD3 Fab groups were combined with two differenttumor targeting Fabs (TAA1 and TAA2) to generate two sets of TCE Biclonics®: CD3xTAA1and CD3xTAA2.

• Three out of four CD3 Fab groups induced TAA dependent cytolysis in combination withTAA1 as well as TAA2

• CD3 Fabs from group 6 demonstrated effective tumor cell killing when combined withTAA1 but not when combined with target TAA2

CONCLUSIONS

Human common light chain (cLC) Fabs binding CD3 were generated by immunization of MeMo®mice with TCR/CD3 receptor complex expressing lipoparticles and T cells and by subsequentpanning of immune phage libraries, constructed from high titer mice. MeMo® is a transgenicmouse containing a human variable heavy chain repertoire and a common human light chain.

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group 1 (0.2289)

group 5 (0.0829)

group 7 (0.0691)

group 6 (0.1765)

group 2 (0.0995)

group 3 (0.0631)

group 4 (0.1423)

group 8 (0.0993)

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